In accordance with well known procedures it is often necessary to determine the cause of a positive direct antiglobulin test performed in the clinical laboratory. Such a determination requires, in part, the isolation of antibodies present on the surface of red blood cells for a determination of their specificity. The process of disassociation of the antibody from the red cell is referred to as elution and the active material for performing this process is the eluting agent. Typically, the antibodies are disassociated from the surface of the red blood cells by the eluting agent and can be subsequently isolated from the eluate.
The collection of antibodies coating the red blood cells in vivo is often essential for the identification and subsequent diagnosis of autoimmune hemolytic anemia, hemolytic disease of the newborn or other suspected hemolytic-type diseases following transfusion reactions. Elution techniques also permit the demonstration of weakly expressed antigens on red blood cells such as those in subgroups of A or B blood types following an in vitro sensitization with an appropriate antibody. Further, elution procedures permit the separation and isolation of individual specificities of antibody mixtures following adsorption in vitro with selected blood cells. Elution procedures are of further utility in the preparation of antisera free of unwanted antibodies following in vitro adsorption with red cells of appropriate phenotype. Elution procedures are further finding increased pharmacological use in the confirmation of sensitization of red blood cells by certain drugs.
The classical method of elution is that of Landsteiner and Miller, described in Blood Transfusion in Clinical Medicine by Mollison, Sixth Edition, January 1979. The Landsteiner et al method simply involved heating the red blood cells to 56.degree. C. in order to remove and recover antibody adsorbed onto the cells at room temperature. Typically, serum was added to the cell suspension and incubated at room temperature in order to sensitive the cells. The cells were then collected by centrifugation, resuspended with saline, and placed in a water bath at 56.degree. C. for five minutes with repeated shaking. Centrifugation of the mixture resulted in a supernatant fluid, or eluate, containing the antibodies. The advantages of this system were numerous; it was inexpensive, easily performed without the requirements of pH adjustments, the eluate remained on top of the cells following centrifugation and a substantial portion of the cell population remained undamaged permitting further elution procedures. Landsteiner's system did, however, require 6% bovine serum albumin and a 56.degree. C. heat source. Further, the method was often incapable of removing for detection antibodies weakly coating the cells. The system also required rapid removal of the eluate in order to prevent potential reassociation of the antibody with available antigenic sites present on the cells.
Subsequent efforts to increase the sensitivity resulted in Rubin's ether elution method, also described in Blood Transfusion in Clinical Medicine, supra. The method required mixing washed and packed red blood cells with an equal volume of 0.8% sodium chloride followed by the addition of two volumes of diethylether in a stoppered container. The container was repeatedly inverted for one minute and then centrifuged for 10 minutes. The top layer of ether was aspirated and the middle layer, containing denatured stroma, was also removed. The bottom layer contained the antibodies of interest. Rubin's ether method successfully provided a good yield as well as an eluate capable of frozen storage in excess of three months without loss of reactivity. The Rubin ether method presented numerous disadvantages however, not the least of which was the use of a potentially explosive compound producing an extreme fire hazard and necessitating explosion proof storage means as well as fume hoods and proper disposal pursuant to OSHA regulations. The procedure further required the careful removal of all residual ether from the final eluate in order to prevent subsequent lysing of reagent red blood cells. Additionally, the great expense associated with the Rubin ether procedure makes this procedure undesirable for large scale clinical testing.
A class of commercially available reagents is known by those in the art as the digitonin/acid method. This method provides for hemolyzation of the red blood cells by digitonin to produce intact stroma free of hemoglobin and capable of ready sedimentation. Typically the stroma is eluted with 0.1 molar glycerin buffer at a pH of three in order to produce a clear eluate. Although the procedure is specific and reliable, it generally requires more steps and larger amounts of reagents. Further, pH correction is critical since any residual reagent may lyse reagent red blood cells subsequently used in specificity testing. The time consuming and complicated nature of the procedure further reduces its effectiveness in clinical applications. R. Bueno, et al presented, in the March-April 1981 edition of Transfusion, an article entitled "Elution of Antibody from Red Blood Cells Using Xylene--A Superior Method". It is claimed that the xylene method was superior to the ether method because it yielded stronger eluates. The method requires the mixture of one volume of sensitized packed red blood cells, one volume of saline, and two volumes of xylene in a stoppered test tube. The mixture is agitated for a minute and then incubated at 57.degree. C. for 10 minutes with occasional mixing. Centrifugation of the mixture at 1000 g for five minutes yields three distinct layers wherein the upper layer containing xylene and the middle layer containing stroma are removed. Additionally, it has been found necessary to remove the top portion of the third eluate layer containing the antibodies. Removal of the stroma layer requires great care in order to avoid contamination of the eluate. An additional disadvantage, characteristic of the xylene method, is the requirement for extensive care in handling since xylene is a toxic aromatic hydrocarbon requiring a fume hood and proper disposal since it presents an explosive hazard. Further, the cost of xylene makes it prohibitive for many routine clinical applications.